Record Information

All applicable rights reserved by the source institution and holding location.

Resource Identifier:

oclc - 25487839ocm25487839

System ID:

AA00011194:00001

Full Text

THE AUTONOMIC NERVOUS SYSTEM: ITS INFLUENCE ON THE
IMMUNE SYSTEM AND DISEASE

By

LEWIS D. FANNON

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1991

ACKNOWLEDGEMENTS

I wish to express my true appreciation to the chairman of my
supervisory committee, Dr. M. Ian Phillips. Dr. Phillips is one of those
rare people who have made a large, positive impact on my life. He has
been a friend as well as a mentor and advisor. Many thanks are also
extended to the members of my supervisory committee. Drs. Colin
Sumners and Mohan Raizada, from the Department of Physiology, have

tried hard to provide me with a positive learning and growth experience
in what has sometimes been a very hostile environment. Special thanks
go to the external member of my committee, Dr. Raul Braylan, from the
Department of Pathology, for introducing me to the techniques used in
this study and for managing to fit me into his busy schedule when it was
needed. Finally, a special note of gratitude goes to Gayle Butters, Birgitta
Kimura and Melissa Chen for the friendship and technical expertise they

H ypothesis..............................................................................................1...
Aim One: To Determine the Effect of
Peptides that Increase Sympathetic
Output on Specific Peripheral
Immune System Components...........................................................2...
Aim Two: To Examine the Role of the
Immune System, the Renin-
Angiotensin System (RAS) and
the Autonomic Nervous System (ANS)
in the Development of Hypertension
in the Spontaneously Hypertensive Rat..........................................6...
Aim Three: To Look at the Relationship
Between the Occurrance of Alterations
in the Immune System and the
Development of Hypertension in
Different Rodent Hypertensive Models.........................................10
Aim Four: The Relationship Between
the ANS and the Development of
Hypertension on Changes Occurring
in the Oral Cavity................................................................................11

2 A METHOD OF MEASURING
THE IMMUNE SYSTEM IN RODENTS........................ 17

Abstract of Dissertation Presented to the Graduate School of the University
of Florida in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy

THE AUTONOMIC NERVOUS SYSTEM: ITS INFLUENCE ON THE
IMMUNE SYSTEM AND DISEASE

By

Lewis D. Fannon

May, 1991

Chairman: M. Ian Phillips
Major Department: Physiology

The premise of this project is that the sympathetic nervous system
(SNS) can act on the immune system to increase susceptibility to disease
states. We were able to quantify different lymphocyte subpopulations in
different experimental and genetic rat models of increased SNS activity
using fluorescence activated cell sorting (FACS) analysis. The lymphocyte
subpopulations examined were the T-cells, B-cells and two T-cell
subgroups, the T-helper cells and the T-nonhelper cells. The initial
experiment involved injecting two peptides, angiotensin II and substance

P, that increase SNS activity, into the brains of intact rodents. The results
showed that the animals receiving the peptide infusions had an increased
percentage of T-cells in their peripheral blood. Another series of
experiments involved two genetic models of increased SNS activity, the
spontaneously hypertensive rat (SHR) and the Dahl S/JR rat. Both of
these strains showed decreases in the percentage of the T-nonhelper
population in the prehypertensive as well as hypertensive phases of
development. Guanethidine treatment of neonatal SHR was undertaken
to destroy the peripheral sympathetic nervous system. These
guanethidine treated SHR had normalized blood pressure and a T-
nonhelper cell percentage in line with the WKY control animals.
Interleukin-2, a powerful T-cell proliferation factor, had no effect on the
blood pressure of the SHR but did increase the T-nonhelper cell percentage
at one time point. Another important finding of this project was the high
levels of angiotensin II, an important neuropeptide and peripheral
vasoconstrictor, found in the spleens of three rat strains. The immune
system has been implicated in the pathogenesis of hypertension in both
man and experimental animals, such as the SHR. In man, another
pathological process, periodontal disease, also is believed to have an
immune component. The mandibles of SHR were found to have
increased bone and tooth loss. The studies indicate that the brain
influences the immune system through the sympathetic nervous system
which can be activated by a brain peptide such as angiotensin II. These
results are relevant to our understanding of the pathogenesis of disease.

CHAPTER 1
INTRODUCTION
Hypothesis
The autonomic nervous system (ANS) regulates and modifies the
immune response, thus altering the susceptibility to certain disease states.
Increased sympathetic nervous system (SNS) activity, such as occurs in
stress situations, leads to alterations in the immune system. These
changes can then result in the development of disease states such as
hypertension or periodontal disease.
The idea that stress affects our health is an old one. It is only relatively
recently that we have been able to obtain experimental evidence
concerning the mechanism behind this observation. Selye (1970) was
among the first to note alterations in the immune system in response to
stress. This thymicolymphatic atrophy was just one of the responses he
observed during the development of his concept of a stereotyped response
to stress. More recently there has been an enormous amount of data
generated linking the central nervous system and the immune system.

The overall aim of our project is to more closely examine the role of the
ANS in regulating and modifying the immune system and then study
how this interaction may be involved in the development of certain
disease states such as hypertension and oral disease. The focus will be on
the actions of the sympathetic portion of the ANS.

2

The following four specific aims provide a structure from which to
examine certain aspects of this question.
Aim One: To Determine the Effect of Peptides that Increase Sympathetic
Output on Specific Peripheral Immune System Components
Angiotensin II (Ang II) has been shown to increase sympathetic output
(Printz and Lewicki, 1977; Aars, 1977; Boadle-Biber and Roth, 1977;
Samuels et al., 1977; Severs et al., 1971; Severs and Daniels-Severs, 1973;
Unger et al., 1981). These studies have involved Ang II in the periphery
and centrally. The peripheral Ang II stimulation of the sympathetic
nervous system (SNS) is brought about mainly by actions on sympathetic
ganglia (Feldberg and Lewis, 1964), adrenal medulla (Feldberg and Lewis,
1964; Reit, 1972) and post-ganglionic enhancement of catecholamine (CA)
release (Zimmerman et al., 1972). Intravenous injection of Ang II has also
been shown to produce an increase in renal nerve activity (Aars, 1977).
Several investigators report an increase in SNS activity to centrally
administered Ang II (Printz and Lewicki, 1977; Aars, 1977; Severs et al.,
1971; Severs and Daniels-Severs, 1973; Unger et al., 1981). The exact
involvement of the various peripheral and central components of the
SNS are not well defined (Unger et al., 1981). Generally, plasma CA levels
are considered a reliable measure of SNS activity (Unger et al., 1981).
When Ang II is given directly into the brain, plasma CA levels increase
(Unger et al., 1981). In addition to this CA effect, it is known that
following intracerebroventricular (ICV) injection of Ang II, arginine
vasopressin (AVP) and adrenocorticotropic hormone (ACTH) levels rise
(Severs and Daniels-Severs, 1973; Reid and Day, 1977). In the brain itself,
centrally injected Ang II appears to stimulate norepinephrine (NE)-rich

nuclei in the brain stem and hypothalamus (Sumners and Phillips, 1983;
Ganten et al., 1980) but does not alter dopamine. Also, Ang II injected ICV
appears to cause the release of NE into the cerebrospinal fluid (CSF)
(Chevillard et al., 1979) and increase CA turnover in the brain stem
(Garcia-Sevilla et al., 1979). Ang II stimulates the release of [3H]-NE
(Schactt, 1984). The peripheral portion of the SNS that mediates the
cardiovascular effects of centrally injected Ang II is also activated but the
relative contribution of this component to the pressor action has not yet
been fully clarified (Unger et al.,1981). There is evidence that AVP release
into the circulation acts along with this peripheral stimulation of the SNS
to participate in the cardiovascular increase to central Ang II (Severs and
.Daniels-Severs, 1973; Unger et al., 1981; Hoffman and Phillips, 1977) and
that these two factors act in parallel. The exact beds which are constricted
by Ang II sympathetic activation are not yet dear although total peripheral
resistance is increased.
Substance P (Sub P) is another peptide which increases sympathetic
outflow when injected centrally. Substance P is a blood pressure
regulating neuropeptide that is localized in brain areas involved in
cardiovascular control such as the medulla oblongata and hypothalamus
(Hokfelt et al., 1978; Gillis et al., 1980). Evidence indicates that there is an
increase in blood pressure following injection into the ventricular system
of the brain (Traczyk and Kubicki, 1980). These Sub P pressor effects are
less potent than those of Ang II, but have a longer duration (Unger et al.,
1981). Following ICV injection of Sub P there appears to be a rise in
plasma NE levels (Unger et al., 1981). This can be considered as an index
of increased sympathetic activity at nerve endings (Yamaguchi and Kopin,

1979) and thus, an indication of an increase in peripheral SNS outflow.
There appears to be no simultaneous increase in AVP as occurs with Ang
II (Unger et al., 1981). There also appears to be some evidence to suggest
that central injection of Sub P causes more sympathetic activation than
seen after central injection of Ang II (Unger et al., 1981). In summation
then, ICV Sub P appears to activate the peripheral portion of the SNS
more intensely than Ang II.
The purpose behind injecting peptides into the brains of intact rodents
is to produce an increase in sympathetic outflow and then measure the
resultant changes in various immune parameters. There is already quite a
large body of evidence suggesting that stress and/or altered ANS activity
can change the immune response and contribute to disease states (Davis
and Jenkins, 1962; DeMarco, 1976; Manhold and Weisinger, 1971; Shields,
1977; Davis, 1984; Workman and La Via, 1987; Marx, 1985). By
experimentally producing an increase in sympathetic activity, it is hoped
that an adequate model will be developed to better study the relationship
between the ANS, the immune response and their interaction in
mediating various disease states not only in animals but in man as well.
This, in turn, will provide more clues to how environment and behavior
influence health. There is already much evidence of this link in the body
as a whole as well as specifically in the oral cavity (Davis and Jenkins, 1962;
DeMarco, 1976; Shields, 1977; Davis, 1984; Workman and La Via, 1987;
Marx, 1985; Haskell, 1975) which will be discussed later. Thus, the
autonomic nervous system may be a mirror of emotional health. It has
been demonstrated that the autonomic nervous system sends fibers to the
thymus gland as well as other immune organs. These nerves tend to
aggregate in areas rich in T-cells and avoid areas where developing B-cells

5

are present (Bulloch, 1985). It has already been shown that an increase in
sympathetic activity is associated with a decrease in immune response
(Cross et al., 1985; Braun et al., 1985; Ader, 1981). It is interesting to note
that a genetically pure animal model exists that demonstrates many of the
previously mentioned connections between altered autonomic activity
and the immune response. The spontaneously hypertensive rat (SHR)
has been shown to have an overactive sympathetic nervous system
(Norman and Dzielak, 1986; Guyton et al., 1974; Iriuchijima, 1973; Judy et
al., 1976; Baird, 1977; Norman and Dzielak, 1982). There are some
provocative data showing that these rats exhibit altered T-cell and B-cell
ratios as well as other, more subtle alterations in their immune systems
indicators of increased brain angiotensin activity (Phillips and Kimura,
1986). This T-cell/B-cell ratio is one of the factors that is important in
maintaining the integrity and proper function of the immune system. It is
our wish to replicate this model experimentally to more closely examine
the mechanism behind and action of this altered autonomic response. By
changing the B-cell and T-cell ratios, many aspects of the immune
response are altered affecting a variety of body functions. An example of
one piece of this puzzle is the action of lymphokines. Lymphokines not
only act locally (Grant et al., 1979a) in the immune response but are
capable of acting centrally as well (Marx, 1985; Blalock, 1984; Blalock et al.,
1982; Smith and Blalock, 1985). These lymphokines, such as ACTH- like
substances (Blalock et al., 1982; Smith and Blalock, 1985) and interleukin,
interferon-like substances (Marx, 1985; Blalock, 1984; Blalock et al., 1979),
are capable of feeding back and acting on various brain areas to further

modulate the autonomic and immune responses. It is interesting to note
that macrophages, which aid in T-cell activation, have been shown to
contain angiotensin converting enzyme (Simon et al., 1986) although it is
unclear exactly how this relates to the renin-angiotensin system's role in
modulating the immune response. These interactions also strengthen the
case for a connection between immune function and CNS function. By
the use of these peptides administered into rat brains, we hope to be able to
better quantify the effect of the ANS on immune components (T-cells, B-
cells, T-cell subsets).
Aim Two: To Examine the Role of the Immune System, the Renin-
Angiotensin System (RAS) and the Autonomic Nervous System (ANS) in
the Development of Hypertension in the Spontaneously Hypertensive Rat
(SHR)
Angiotensin converting enzyme (ACE) inhibitors are used clinically for
the reduction of blood pressure, but their exact mechanism of action is not
yet fully understood (Unger et al., 1983). It has been shown that chronic
administration of ACE inhibitors lowers ACE activity levels in brain, aorta
and kidney. The action of ACE inhibitors on lung and plasma ACE
activity has not been as clear (Ikemoto et al., 1986; Moursi et al., 1986).
Plasma renin activity increases with age in stroke prone spontaneously
hypertensive rats (SHR-SP) (Matsunaga et al., 1975) and angiotensin II
levels in the brain of the SHR are higher than control values (Phillips and
Kimura, 1986). Along these same lines, it is interesting to note that
Wilson et al. (1988) have shown that chronic treatment with the ACE
inhibitor Captopril will reduce Ang II receptor binding in the brain of the
SHR. Norman and Dzielak (1986), as well as others (Judy et al., 1976), have
demonstrated that the SHR's sympathetic nervous system activity is

increased relative to controls. Several experiments have shown that ACE
inhibitors will reduce hypertension in the SHR (Ikemoto et al., 1986;

Moursi et al., 1986; Cohen and Kurz, 1982; Unger et al., 1985), but the exact
mechanism of this action is still the topic of much debate.
The spleen is an important organ in the immune system. It filters
blood in much the same way as lymph nodes filter lymph. During an
episode of infection the spleen is one of the sites of lymphocyte
proliferation and differentiation. Distinct populations of macrophages
and other immunocompetent cells are also found in the spleen. Castren
et al. (1987) have shown that there is considerable binding of Ang II in the
spleen. This binding tends to be localized in the red pulp of this organ.
Ang II binding has been found on lymphocytes (Shimada and Yazaki,
1978) and macrophages (Thomas and Hoffman, 1984; Weinstock and
Kassab, 1984). Ang II has an inhibitory action on leukocytes probably
caused by a stimulation of T-suppressor cells (Simon et al., 1986).
Macrophages also contain angiotensin converting enzyme, indicating that
Ang II may have significance in immune function (Hinman et al., 1979).
Given this information, it would be useful to determine what levels of
Ang II exist in the spleen.
Immune abnormalities have been suggested as a causative factor in
both oral disease and hypertension. The connection between
hypertension and immune abnormalities in both humans and
spontaneously hypertensive rodents has been repeatedly documented
(Norman and Dzielak, 1986; Khraibe et al., 1984; Bendich et al., 1981;
Svendsen, 1979; Munro, 1978; Kirstensen, 1979, Olsen et al., 1973). The
SHR exhibits an increased sympathetic nerve activity (Norman and
Dzielak, 1986; Judy et al., 1976) and several investigators have linked this

to changes in the immune system (Norman and Dzielak, 1986; Judy et al.,
1976; Takeichi et al., 1986; Khraibe et al., 1984; Takeichi et al., 1981;
Fernandes et al., 1986). These changes involve alterations in the B-cell, T-
cell ratio which appears to be significant in the pathogenesis of
hypertension (Bendich et al., 1981). The T-suppressor cells in the SHR are
especially depressed (Norman and Dzielak, 1986). However, no data are
available as to when these changes occur and how they relate to the
development of hypertension. We intend to look at these lymphocyte
populations in both the prehypertensive and hypertensive phases of
development. The T-suppressor cells are an important regulator of
lymphokine secretion. B-lymphocytes, like T-lymphocytes, are capable of
lymphokine secretion (Grant et al., 1979a). Lymphocytes can also secrete as
a lymphokine an interferon-like substance as well as an ACTH-like
substance that will cause adrenal cortisol secretion (Blalock, 1984). The
role of the altered B-cell, T-cell ratio and resultant changes in lymphokine
secretion in the SHR is unclear. The immunologic defects in the SHR in
many ways parallel those found in human hypertensive conditions
(Norman and Dzielak, 1986; Svendsen, 1979; Munro, 1978; Kirstensen,
1979; Olsen et al., 1973). These defects may trigger hypertension by causing
vascular inflammation (Suzuki et al., 1978) or renal glomerular damage
(Evan et al., 1981). The increased sympathetic activity may also play a role
by increasing circulating catecholamines (Borkowski and Quinn, 1984).
Another possible mechanism may involve direct action of lymphokines
on the CNS and periphery.
A recent study by Tuttle and Boppana (1990) showed that when
interleukin-2 (IL-2) is given to prehypertensive SHR the development of

hypertension is abolished. A decrease in blood pressure was also seen in
adult SHR given IL-2. IL-2 is released by stimulated T-lymphocytes and

causes the proliferation of activated T-cells. Both T-helper cells and
cytotoxic T-cells are affected. The results of their experiment are
strengthened by data from others showing that there is an impairment
and depression of T-lymphocyte function in the SHR (Takeichi et al., 1986;
Takeichi et al., 1981). Also transplants of thymic tissue from Wistar Kyoto
(WKY) rats into neonatal SHR can attenuate the development of
hypertension (Norman and Dzielak, 1986). The thymus gland is involved
in the development of T-cells so there is a precedent for this finding that
IL-2, a strong T-cell activating factor, could affect the development of
,hypertension in this model. A goal of this project is to repeat this study
and see if it affects the proportions of the different lymphocyte
populations.
In order to more fully explore the influence of the ANS, specifically the
SNS, on immune activity it would be beneficial to block the SNS and
examine any resultant changes in the immune system. Guanethidine,
when given from birth produces a peripheral sympathectomy (Johnson
et al., 1975). We intend to use this as an experimental model of reduced
SNS activity and then measure any resultant changes in immune
parameters. As has been mentioned previously, there is much evidence
to suggest that SNS innervation to immune organs can alter immune cell
ratios as well as immune function. SHR exhibit increased sympathetic
activity as well as altered immune characteristics. By blocking this activity
with guanethidine a better understanding of the ANS and its regulation of
immune function can be obtained.

Aim Three: To Look at the Relationship Between the Occurance of
Alterations in the Immune System and the Development of
Hypertension in Different Rodent Hypertensive Models
It has already been shown that there is evidence of a link between the
immune system and the development of hypertension in humans
(Svendsen, 1979; Munro, 1978; Kirstensen, 1979; Olsen et al., 1973) as well
as animals such the SHR (Norman and Dzielak, 1986; Unger et al., 1985;
Khraibe et al., 1984; Bendich et al., 1981). In order to more carefully look at
the immune system as one of the causative factors in hypertension,
different hypertensive models should be examined. Other rodent models
of hypertension have been left largely unexplored. The inbred Dahl rat
strains are another genetic model of hypertension (Rapp and Dene, 1985).
"The original strain produced by Dahl was found not to be genetically pure.
From the original Dahl strain, however, a genetically pure form has been
developed (Rapp and Dene, 1985). The two strains, the Dahl salt sensitive
(S/JR) and the Dahl salt resistant (R/JR), show different susceptiblities to
sodium induced hypertension. When fed a high sodium diet, the S/JR
rats will develop a severe hypertension that does not develop in the R/JR
animals fed the same diet. Importantly, the S/JR will develop
hypertension when on a normal sodium diet although not as rapidly
(Rapp and Dene, 1985). These animals exhibit alterations in their CA
usage (Kuchel et al., 1987; Racz et al., 1987) and also show the same type of
vascular inflammation as that seen in the SHR (Rapp and Dene, 1985;
Suzuki et al., 1978). By examining any immune system alterations that
may occur in these animals, it is possible that a better understanding of
their hypertension as well as of hypertension in general will develop.

Aim Four: The Relationship Between the ANS and the Development of
Hypertension on Changes Occuring in the Oral Cavity
A further aim of our study is to look at the direct effects of the
autonomic nervous system and the development of hypertension on the
oral cavity in order to better understand the relationship and
characteristics of the involved mechanisms. Alveolar bone and tooth loss
are the parameters that will be examined. Although many studies have
looked at the hypertensive characteristics of the SHR, we have not been
able to find any work which has been done on the oral cavities of the SHR.
As has been stated previously, the SHR has an overactive sympathetic
nervous system. Normally, most blood vessels, including those of the
periodontium, or supporting structures of the tooth, are controlled by
sympathetic vascular tone with the tissue cells themselves playing a role
via local humoral factors (Grant et al., 1979a). In the case of the chronic
inflammatory process associated with the majority of periodontal diseases,
these normal control mechanisms are again in operation, but to differing
extents with their exact role not as clearly understood. The typical
inflammatory response, including that associated with periodontal
disease, can be initiated by both cellular and humoral immunologic
reactions (Hyman and Zeldow, 1963; Rizzo and Mitchell, 1966; Ranney and
Zander, 1970) as well as directly by bacterial endotoxins and enzymes
(Grant et al., 1979a). Both of these processes can also activate the
complement system which is capable of inducing inflammatory effects on
its own (Grant et al., 1979a; Mergenhagen, 1970). Inflammation begins
with a transient vasoconstriction followed by a prolonged vasodilation
(Bhaskar, 1977; Rocha de silva and Leme, 1972; Zweifach, 1973). Following
vasodilation, blood flow at first quickens, then slows to a more static pace

because of 1) increased viscosity of blood due to a loss of fluid from the
vascular compartment (brought about by an increase in hydrostatic
pressure produced by a lag in venular dilation) and 2) a vascular
permeability increase due to the presence of protease systems (Spragg, 1974;
Erdos, 1968) and lymphokines (Grant et al., 1979a). During the late phase
of the inflammatory process, sludging and thrombus formation may occur
from fibrin formation brought about by platelet aggregation. If severe
enough, these vascular changes may bring about ischemia, tissue anoxia,
acidosis and necrosis of the involved area. In the normal case, following
this defense mechanism the body attempts to affect repair by fibroblastic
proliferation and new capillary formation into the affected area. This
reparativee process necessitates an increased oxygen uptake in the involved
tissues. If the conditions that initiated the inflammatory response
continue and/or there is insufficient tissue oxygen for repair to take place,
a chronic inflammatory condition may ensue which, in the case of
periodontal tissue, results in many of the pathological characteristics
associated with periodontal disease, i.e. soft tissue and bone destruction.
Thus, any process that blocks or inhibits periodontal blood flow, and thus
oxygen availability, may play a role in periodontal pathology (Manhold
and Weisinger, 1971).
The connection between stress and oral pathology has been well
established in both human and animal studies (Manhold and Manhold,
1949; Goldberg et al., 1956; Miller et al., 1956; Moulton et al., 1952; Pierce,
1960; Giddon et al., 1962; Baker et al., 1961; Belting and Gupta, 1960;
Manhold, 1958; Fedi, 1958; Gupta et al., 1960; Steinman, 1960; Hollomond,
1962; Steinman et al., 1961; Liu and Liu, 1969). It is the mechanism that

has remained somewhat elusive. Manhold, (1956), was among the first to
propose a connection between the autonomic nervous system and
periodontal pathology. He hypothesized a model in which increased
sympathetic activity could result in a constriction of blood vessels and
thus, a lack of oxygen and nutrients to the periodontium (Manhold and
Weisinger, 1971; Manhold, 1956). This would be especially important
during the initial reparative stages following an inflammatory episode
and could lead to a pathological periodontal breakdown (Manhold and
Weisinger, 1971) via a progressive, chronic inflammatory response which
is unable to resolve itself. Thus, the relationship between oxygen
utilization and oral health is an important one (Glickman et al., 1949). As
the tissue progresses from a healthy state to a diseased state, the oxygen
utilization has been experimentally shown to change. A chronic marginal
gingivitis is associated with an increased 02 utilization brought about by
the formation of new blood vessels and connective tissue (Glickman et al.,
1949) as the inflamed tissue attempts to heal itself. It is at this point that a
restriction of oxygen and nutrients could lead to a progressive chronic
inflammation and periodontal pathology. A restriction of 02 would also
favor colonization by the anaerobic bacteria associated with periodontal
pathology (Grant et al., 1979b). A direct sympathetic activity in the oral
cavity may provide an explanation for this pattern of progression and
might help explain why people with similar oral hygiene characteristics
have varying degrees of periodontal disease. Degenerating tissue
(Glickman et al., 1949) and the tissue of animals subjected to chronic stress
(Manhold and Weisinger, 1971) show a marked decrease in gingival
oxygen utilization. A lack of oxygen during this crucial initial stage of

repair brought about by an unbalanced autonomic activity may play a role
in the spread and progression of the inflammatory response.
It is also possible that autonomic induced alterations in the immune
system may play a role. The immune system has also been implicated in
many aspects of periodontal disease (Grant et al., 1979a, Nisengard, 1977).
Although a link between these immune abnormalities and the autonomic
nervous system has not been firmly established, there has been a
connection made between autonomic activity and periodontal disease
(Davis and Jenkins, 1962; Manhold and Weisinger, 1971). As the condition
progresses the number of B-cells increase relative to T-cells (Grant et al.,
1979a). As has been pointed out earlier, these B-cells are capable of
producing lymphokines whose physiochemical and/or biologic activities
are similar to those derived from T-cells such as migration inhibitory
factor (MIF) (Yoshida et al., 1973) and osteoclast activating factor (OAF)
(Mundy et al., 1974). Although it has not been experimentally
demonstrated, it is reasonable to assume that the lymphocytes involved in
periodontal pathology are also capable of releasing ACTH-like and
interferon-like lymphokines, which are capable of acting both locally and
peripherally. In order to examine the connection between autonomic
activity, immune characteristics and oral disease, an adequate model
should be used. The oral characteristics of the SHR which possesses an
inherent sympathetic overactivity and resultant immune abnormalities
have never been examined.
Very little work has been done to suggest a common etiology of
hypertension and periodontal disease. Although much is known
concerning the relationship between stress and the characteristics of
advanced hypertension, it is only relatively recently that any focus has

been given to an autonomic-immune component (Norman and Dzielak,
1986). The same can be said for periodontal disease. Here, the main focus
has been in the area of microbiology, bacteriology and their relationship to
the advanced stages of the disease. A relatively small emphasis has been
given to those peripheral factors which allowed the bacterial changes and
proliferation to occur in the first place. Once again, although a connection
has been shown between stress and oral disease (Davis and Jenkins, 1962;
DeMarco, 1976; Manhold and Weisinger, 1971; Shields, 1977), it is only
quite recently that an autonomic-immune component to the etiology has
been considered. Current treatment modalities for both hypertension and
periodontal disease are often aimed at the end results of the disease
process rather than the initial mechanism. By examining the
characteristics of both disease processes, a common etiology and hence,
better understanding, can be accomplished.
A possible simplified mechanism of action may be initiated by a chronic
stress (bacterial invasion, social stress, emotional and physiological
disharmony) situation. In a chronic stress situation, an adaptation
response will occur for cortisol and the increased levels will eventually
diminish. This is not the case with catecholamines. Upon repeated
stresses, the catecholamine levels will still increase (Rose, 1980). This
stress can induce activation of the sympathetic nervous system (SNS) to
the oral cavity as well as peripheral immune organs. This decreases the
number of T-cells, especially T-suppressor cells (Norman and Dzielak,
1986), relative to B-cells and thus, removes control of B-cell and remaining
T-cell lymphokine secretion provided by the T-suppressor cells. The
unchecked B-cells and remaining T-cells are then able to increase secretion

16

of lymphokines such as ACTH-like substances, interferon-like substances,
as well as others (OAF, MIF, etc.) which can act locally in the mouth as
well as directly upon the hypothalamus and other structures to further
potentiate ANS activity. During certain stress situations, it may be
lymphocyte derived ACTH rather than pituitary derived ACTH that
mediates increased cortisol secretion (Blalock, 1984; Blalock et al., 1982).

CHAPTER 2
A METHOD OF MEASURING THE IMMUNE SYSTEM IN RODENTS

Introduction
Over the past several years there has been an explosion of studies in the
field of psychoneuroimmunology. This field attempts to examine how
the brain and behavior can influence health and the susceptiblilty to
disease through the immune system. Lymphocytes are a major effector
cell in the immune system. There are two major types, T-lymphocytes
and B-lymphocytes. T-cells are lymphocytes which develop in the
thymus. The thymus is seeded during embryonic development by stem
cells from the bone marrow (Male, 1986a). There is a considerable amount
of T-cell proliferation and death in the thymus. Most lymphocytes will die
before ever leaving the thymus. During their development in the
thymus, T-cells acquire their antigen receptors and differentiate into
distinct subpopulations. One of these subpopulations is the T-nonhelper
cells. This population includes the T-cytotoxic cells which are capable of

destroying altered "self" cells and also virally infected "self" cells. The T-
nonhelper population also includes the T-suppressor cells which regulate
the action of other T-cells and B-cells. Their suppressive action can be
either specific for cells with particular antigen receptors or non-specific
(Male, 1986a). Another distinct population of T-cells are the T-helper cells.
These cells release lymphokines which can activate macrophages and
other T-cells as well as help B-cells to produce antibody (Male, 1986a). B-

cells are lymphocytes which develop in the bone marrow. B-cells that are
stimulated by lymphokines released by T-cells, differentiate into plasma
cells that are antibody producing cells. These cell to cell interactions are
summarized in Figure 2-1. The chemical mediators released by all these
different cell types can act locally at tissue sites of immune system activity
and directly or indirectly feed back on the CNS.

There are several different ways of measuring the activity of the
immune system, natural killer (NK) cell activity, spleen cell "panning"
(Wysocki and Sato, 1978), thymus weights, IgG levels, but to measure the

immune status accurately, the blood concentrations of lymphocytes and
proportions of B-cells to T-cells needs to be measured. The method
*described here was developed specifically for blood lymphocyte
measurement in the rat. The percentages of T-cells, T-nonhelper cells, T-
helper and B-cells are given by this method. It is accurate and rapid.

Methods
Blood Sampling

The rats are anesthetized with Metofane anesthesia (Pitman-Moore,
Inc., Washington Crossing, NJ). The area of skin covering the left femoral
vein was prepared and cleaned with a 70% ethanol solution. A two
centimeter incision was made and the femoral vein exposed using minor
dissection. Using a 25 gauge, butterfly scalp vein infusion set (Abbott
Hospitals, Inc., North Chicago, IL) venipuncture was performed and one
milliliter (ml) of blood obtained. A cotton swab was used to apply direct
pressure to the vessel upon removal of the needle. Wound clips were
used to close the incision and the blood was placed in sterile vacutainer
tubes (Becton-Dickinson, Rutherford, NJ). This method is a fast and

efficient mode of blood collection as well as venous drug delivery. It is
also useful when repeated samples are needed from the same animal over
time, as the same incision and vessel can be used. With practice, blood
samples can be collected from seven to ten animals in an hour. Also, this
method has many advantages over venous catheterization.
Lymphocyte Preparation
The one ml whole blood sample was combined with one ml of PBS
with 0.1% NaN3 (pH = 7.4) that had been chilled on ice. This solution was
layered on top of three ml of Lymphocyte Separation Medium (LSM,
Organon-Teknika, Durham, NC) in a 14 ml conical tube that had been
lightly coated with fetal bovine serum (FBS). The tubes were centrifuged
at 400G for 30 minutes at 120C. Following centrifugation, the white layer
found between the plasma and LSM is predominantly composed of
lymphocytes. This layer was aspirated and placed in a tube coated with
FBS and containing one ml of chilled PBS with 0.1% NaN3. All
subsequent tubes were lightly coated with FBS to prevent adhesion of cells
to the surface of the plastic tubes. The lymphocyte suspension was washed
twice with chilled PBS with 0.1% NaN3 prior to staining with monoclonal
antibodies. The cells are washed by adding the PBS and centrifugating at
400g for 5 minutes. This is followed by aspiration of most of the
supernatant, vortexing the pellet and the addition of the next 3 ml of
chilled PBS.
Four lymphocyte populations were examined: T-cells, T-nonhelper
cells, T-helper cells and B-cells. Mouse anti-rat monoclonal antibodies
(Accurate Chemical, Westbury, NY) were used as primary antibodies and a
goat anti-mouse FITC conjugated reagent (Accurate Chemical) was used as
a secondary antibody. As a control, mouse IgG was used.

The specificity of each monoclonal antibody was provided by the
company as follows: Clone W3/13 HLK (T-cells) labels all thymocytes and
peripheral T-lymphocytes, but not B-lymphocytes; antigen also in brain.
Clone OX8 (T-nonhelper cells) labels most thymocytes and the peripheral
T-cell subset which includes cytotoxic and suppressor T-cells. The T-
helper subset is not labelled. Some natural killer cells are also labelled.
Clone W3/25 (T-helper cells) labels most thymocytes and the helper T-
lymphocyte subset. No other cells are known to be recognized. Clone
OX33 (B-cells) recognizes that portion of the rat leucocyte common antigen
seen on B-lymphocytes. The secondary antibody used was a fluorescein
conjugated goat anti-mouse IgG produced to have minimal cross reaction
*to human, bovine, horse and rat serum proteins.
For each sample approximately 1.5 x 106 cells in a 300 microliter (ul)
volume were placed in each of five, labelled, 12 x 75, clear plastic tubes.
The tubes were labelled as follows: control, T-cell, T-nonhelper, T-helper
and B-cell. The appropriate concentration of mouse IgG or monoclonal
antibody was added to each tube and the tubes were incubated for 30
minutes at four degrees centigrade. Our lab has obtained good results by
adding three ul of the stock antibody solution (the ascites fluid form) to
the 300 ul lymphocyte suspension. Following this initial incubation, the
cells were washed three times with three ml of PBS with 0.1% NaN3.
After the third wash, the tubes were aspirated to approximately 300 ul
and then 100 ul of a 1:100 dilution of the secondary FITC labelled antibody
was placed in each tube. This should be done in a room with the
overhead lights turned off and considerable care should be exercised in the
remaining steps to protect the samples from as much light as possible.

The cells were again incubated for 30 minutes at four degrees centigrade.
After an additional three washes with three ml of PBS with 0.1% NaN3,
each tube was adjusted to a final volume of approximately 500 ul prior to
fluorescence activated cell sorting (FACS) analysis. A summary of these
steps in shown in Figure 2-2.

Results
Fluorescence activated cell sorting analysis involves hitting a thin
stream of the cell suspension with a laser and then utilizing light detectors
to collect the scattered light. All samples were run on a FACStar Plus
(Becton-Dickinson, Rutherford, NJ). Information regarding cell size and
granularity can be obtained and utilized to selectively study a single
-population of cells, such as lymphocytes in this case (Figure 2-3). Side
scatter is an indication of cell granularity while forward scatter tends to be
an indication of cell size. As is seen in Figure 2-4, fluorescently labelled
cells can also be quantified by this method. Background fluorescence can
be deleted and the percentage of labelled cells quantified.
We have shown here a method for identifying and quantifying
different lymphocyte populations in rats. The method is quick, efficient
and reliable. It is also highly reproducible. This method provides another
tool for looking at the immune system during a variety of experimental
conditions.

/ Final cell suspension brought
Approximately 1.5 million to 5 for FACS analysis
cells are placed in each of
five labelled tubes
(control, T-cell, T-nonhelper,
T-helper and B-cell).

Figure 2-2: Summary of lymphocyte preparation steps.

ssc MC
A B

Figure 2-3: Fluorescence activated cell sorting of rat blood. Side scatter is
an indication of cell granularity, while forward scatter tends ot be an
indication of cell size. A: Whole blood that has had red blood cells lysed.
Populations of lymphocytes, monocytes and macrophages can be seen. B:
Whole blood that was separated using Lymphocyte Separation Medium.
Note the relative purity of the lymphocyte population. In both cases the
lymphocyte population has been boxed in.

CHAPTER 3
CHRONIC ICV INFUSION OF NEUROPEPTIDES ALTERS
LYMPHOCYTE POPULATIONS IN EXPERIMENTAL RODENTS
Introduction
A cause and effect relationship between emotional state and
susceptibility to disease has long been suspected. From this cause and
effect relationship the emphasis has now shifted to the examination of
possible mechanisms that are involved. Peptides in the brain are being
investigated for their role in controlling the immune system.
Corticotropin releasing factor (CRF), arginine vasopressin (AVP),
Substance P (Sub P), neurotensin, vasoactive intestinal polypeptide (VIP),
somatostatin and opiod peptides are examples of neuropeptides with
reported immunomodulatory properties (Yirmiya et al., 1989; Morley and
Kay, 1986). Corticotropin releasing factor is a peptide linked to neural
responses of stress; central administration of CRF has been studied in
relation to its effects on the sympathetic nervous system (SNS) (Brown et
al., 1982) and the immune system (Irwin et al., 1988). It appears that the
effect of CRF is to decrease the natural killer cell activity. This has been
shown to be through an action on the SNS (Irwin et al., 1988). Several
studies have looked at the SNS and its influence on the immune system
(Ader, 1981; Braun et al., 1985). The general hypothesis is that stressors
induce elevation of the autonomic sympathetic nervous system which
compromises the immune system and increases the chance of

opportunistic infection or reduced antitumor resistance. Angiotensin II
and substance P are two other important neuropeptides that have shown
the ability to increase SNS activity. These peptides may act in the same
way as CRF to alter different lymphocyte populations.

When delivered into the lateral brain ventricles, Ang II has been
shown to produce an increase in sympathetic output, as indicated by an
increase in plasma norepinephrine levels (Unger et al., 1981). Other
phenomena associated with intracerebroventricular (ICV) Ang II infusion
include an increase in blood pressure and drinking as well as an increase
in AVP secretion (Phillips, 1987). Administration of Ang II to the brain
has also been associated with the release of ACTH (Reid and Day, 1977;
Maran and Yates, 1977). Sub P also has been shown to cause an increase in
SNS activity but does not show the increase in AVP secretion
characteristic of central Ang II infusion (Unger et al., 1981). The effect of
centrally administered Sub P on ACTH release is unknown. When
injected centrally, Sub P seems to be more of a pure SNS activator .
In the present study we chronically infused Ang II and Sub P into the
brains of intact rodents for one month and two weeks respectively.
Following this chronic administration, several populations of
lymphocytes were examined in order to further understand the effect of
these centrally active peptides on the immune system.
Purpose
This study was to examine the effect of peptides that increase SNS
activity on the percentage of the different lymphocyte populations that
were of interest.

Rationale
The sympathetic nervous system has been shown to influence the
immune system. Specifically, an increase in SNS activity is associated
with a decrease in immune activity.

Methods
Animals
Four to five month old, male, Sprague-Dawley rats were obtained from
Charles River Laboratories (Wilmington, MA). Upon arrival all animals
were placed in a room with a 12:12 light/dark cycle with ad lib rat chow
(Purina Mills, St. Louis, MO). Water intake was measured using tap water
in graduated drinking flasks.
.Implantation of Osmotic Minipumps
Angiotensin II (Sigma Chemical, St. Louis, MO) was to be delivered into
the lateral brain ventricle for a chronic infusion of one month via osmotic
minipumps. In preparation for surgery, the animals were anesthetized
with 5% chloral hydrate in saline (0.8 ml per 100g body weight). A shaved
area of skin over the skull to the middle of the back was washed with a
betadine solution, followed by 70% ethyl alcohol. Two incisions were
made; intrascapularly and on the skull. The tissue covering the skull was
pushed away until the bregma could be identified.
A 22 gauge, L-shaped cannula (Small Parts, Inc., Miami, FL) was
attached to the filled osmotic minipumps via non-kinking vinyl tubing
(Bolab, Inc., Lake Havasu, AZ). The cannula was fed under the skin from
the intrascapular incision to the skull incision. The cannula was then
stereotaxically placed at L=1.0mm, P=1.0mm, V=-5.0mm with respect to

Bregma. Methyl methacrylate cement was used to attach the cannulae to
previously placed retention screws.
Alzet osmotic minipumps (Alza Corp., Palo Alto, CA) were used for
drug delivery. The model 2002 pumps deliver at a rate of 0.5 Wl per hour
for 14 days. A sterile Ang II solution was prepared to a concentration that
would allow for a one ug/hr release of Ang II over the 14 day life of the
pump. The Ang II was dissolved in a vehicle of artificial cerebrospinal
fluid (CSF), the formula for which was supplied by the Alza Corporation.
Angiotensin II has been used in several studies involving Alzet
osmotic minipumps. We had some question, however, regarding the
stability of the Ang H over the entire functional life of the pump. We
placed several model 2002 pumps in flasks containing normal saline and
then placed these flasks in a water bath at 370C for 14 days. All pumps
contained Ang II dissolved in artificial CSF. Using radioimmunoassay, we
found that immunoreactive Ang II was being delivered by the pumps at
the rate stated by the company.
As the model 2002 pump is only used for a 14 day period, the Ang II
containing pumps were replaced on day 14. Metofane anesthesia (Pitman-
Moore, Inc., Washington Crossing, NJ) was used for this brief procedure.
Substance P was to be delivered into the lateral brain ventricle for a
chronic infusion of two weeks. The procedure was the same as that for
Ang II except that the Sub P was dissolved in the artificial CSF to a
concentration that would allow for a two ug/hr release of Sub P over the
14 day life of the pump.

Drinking Measurements
Only animals that showed an increase in drinking, relative to controls,
were utilized as Ang II infused animals.
Blood Pressure Measurements
Systolic blood pressure was measured in the Sub P rats on day 12 using a
tail plethysmograph.
Lymphocyte Preparation
One milliter of whole blood was obtained from the left femoral vein
under metofane anesthesia as described in Chapter 2. Lymphocytes were
separated and prepared for FACS analysis as described in Chapter 2.
Statistics
Student's t test was used to compare the means of the two groups for
each experimental protocol (Ang II infusion or Sub P infusion). A
significance level of 0.05 was used.

Results
Angiotensin II is a powerful dipsogen when injected directly into the
brain ventricles. The effect is so reliable that a measurement of drinking is
a valid measure of cannula patency in the brain ventricles. Fig. 3-1 shows

that the constant infusion of Ang 1 via the minipumps was effective as
illustrated by the increase in drinking by the experimental group.
Figure 3-2 shows graphically the results of quantifying the percentage of
different lymphocyte populations. As can be seen, there was a significant
increase in the percentage of T-cells (84.53.1 vs 74.12.3) and a decrease in
the percentage of B-cells (14.21.6 vs 20.30.9), present in the Ang II
infused rats, relative to the artificial CSF infused controls. In the Sub P
infused rats, there was also a significant increase in the percentage of T-
cells relative to the control animals (83.83.4 vs 74.31.4) (Figure 3-3). The

Sub P infused animals, however, did not show the decrease in the
percentage of B-cells that was seen in the Ang II animals. No significant
differences were noted in the T-nonhelper or T-helper populations of
either experimental group.
Acute Sub P injections into the brain ventricles have been shown to
increase blood pressure (Unger et al., 1981). Although the Sub P infused
animals showed a trend toward higher blood pressure after 12 days
relative to the artificial CSF control animals, the groups were not
statistically different (Table 3-1).

Discussion
Our goal was to look at two experimental models of centrally induced
'increased SNS activity and see if this resulted in any alteration in the
percentage of different lymphocyte populations. Other measures of the
immune system have been used to show that CRF alters immune
competence (Irwin et al., 1988). This effect is due to sympathetic action on
the immune system. Centrally infused Ang II has been shown to cause an
increase in sympathetic nervous system activity (Unger et al., 1981;
Phillips, 1987) as has Sub P (Unger et al., 1981). Therefore, these changes
are most likely due in part to this increased SNS activity.
It is interesting to speculate on why the Ang II infused animals showed
a decrease in B-cells and why the Sub P animals did not. As has been
mentioned previously, Ang II infused into the brains of intact rodents
causes not only an increase in SNS activity, but also an increase in AVP
release (Unger et al., 1981; Phillips, 1987) and ACTH release (Reid and Day,
1977; Maran and Yates, 1977). AVP has been suggested to play a role in
immunoregulation (Yirmiya et al., 1989). AVP immunoreactivity has been

found in such tissues as lymph nodes (Aravich et al., 1987) and thymus
(Markwick et al, 1986). AVP receptors are found on lymphocytes and these
receptors are thought to play a role in lymphokine production (Johnson
and Torres, 1985). The possibility exists that AVP may be acting on certain
lymphocyte populations, such as the T-helper population, to influence the
production of B-cell sensitive lymphokines. ACTH also has
immunoregulatory properties through its action on corticosteroid release.
Macrophages are particularly sensitive as corticosteroids can inhibit their
activation. Lymphocytes are also effected. Corticosteroids inhibit the
primary antibody response and reduce the numbers of circulating T-cells,
especially T-helper cells (Male, 1986b). The data from this experiment,
'however, do not indicate a reduction in any T-cell population examined.
In fact, an increase in the percentage of total T-cells was observed.
Also of interest is the comparison of this work with experiments done
in our lab on genetic models of increased sympathetic nervous system
activity. The spontaneously hypertensive rat (SHR) has been shown to
have an increased SNS activity that is at least in part responsible for the
hypertensive state (Norman and Dzielak, 1986; Judy et al., 1976). We have
shown that adult SHR demonstrate a decrease in the percentage of
lymphocytes from each of the populations studied (Chapter 4). Although
both the genetic and experimental models have an increased SNS activity,
dearly other factors are at work here.
By changing the B-cell and T-cell ratios, many aspects of the immune
response are altered affecting a variety of body functions. SHR exhibit
increased brain levels of Ang II (Phillips and Kimura, 1988) and increased
sympathetic nerve activity (Norman and Dzielak, 1986; Judy et al., 1976).

Several investigators have suggested changes in the immune system as at
least a partial cause of the hypertensive state (Takeichi et al., 1986; Khraibe
et al., 1984; Takeichi et al., 1981; Fernandes et al., 1986) in this model.
These changes involve alterations in the B-cell, T-cell ratio which appears
to be significant in the pathogenesis of hypertension (Khraibe et al., 1984;
Bendich et al., 1981). Bulloch (1985) has shown that the ANS sends fibers
to the immune organs. These fibers richly innervate areas high in T-cell
concentration and avoid areas that contain developing B-cells.
Based upon this information a possible simplified mechanism of
action may be hypothesized. In a chronic stress (bacterial invasion, social
stress, emotional and physiological disharmony) situation, an adaption
response will occur for cortisol and the increased levels will eventually
diminish. This is not the case with catecholamines. Upon repeated
stresses, the catecholamine levels will still increase (Rose, 1980). This
stress can induce activation of the sympathetic nervous system (SNS) to
various parts of the body, including peripheral immune organs. This
decreases the number of T-cells, especially T-suppressor cells, relative to B-
cells and thus, removes control of B-cell and remaining T-cell lymphokine
secretion provided by the T-suppressor cells. The unchecked B-cells and
remaining T-cells are then able to increase secretion of lymphokines such
as ACTH-like substances, IFN-like substances, as well as others (OAF, MIF,
etc.) which can act locally in tissues, as well as directly upon the
hypothalamus and other structures to further potentiate ANS activity. It
is the B-cell to T-cell ratio that is important. This view is supported by the
fact that during certain stress situations, it may be lymphocyte derived
ACTH rather than pituitary derived ACTH that mediates increased
cortisol secretion (Blalock et al., 1982; Blalock, 1984).

In summary, we chronically infused neuropeptides into the brains of
intact rodents. We found alterations in the percentages of different
lymphocyte populations. Specifically, the Ang II infused animals
demonstrated a decrease in the percentage of B-cells and an increase in the
number of T-cells. The Sub P infused animals also exhibited this increase
in T-cells, but failed to show any alteration in the percentage of B-cells.
Finally, this study adds to the growing body of data suggesting an
important role of the CNS in regulating immune function and
susceptibility to disease.

100

90-
Ang II (n=5)
80 Control (n=5)

70-

g 60 -

50

40
0
.9 30
V-4
20

10 1 *, I I I
Day 0 Day 8 Day 13 Day 18 Day 21 Day 24 Day 28

Figure 3-1: Drinking response to chronic ICV Ang II infusion via osmotic
minipumps in Ang II infused rats versus artificial CSF infused control
rats. Water intake was measured as total amount consumed when water
bottles were available for 12 hours. All values are expressed as Mean
SEM.

CHAPTER 4
ALTERATIONS OF LYMPHOCYTE POPULATIONS DURING
DEVELOPMENT IN THE SPONTANEOUSLY HYPERTENSIVE RAT
Introduction
The spontaneously hypertensive rat (SHR) is widely used as one of the
models for human essential hypertension. The causes of this
hypertensive state are not yet fully understood and many possibilities are
being investigated. Among the factors considered are alterations in the
central renin-angiotensin system (Phillips and Kimura, 1988), increased
sympathetic nervous system (SNS) activity (Norman and Dzielak, 1986;
Judy et al., 1976) and increased arginine vasopressin (AVP) secretion
(Crofton et al., 1978). Some evidence is accumulating to lend support to
the idea that the immune system is also involved in the development of
hypertension in the SHR (Norman et al., 1985; Norman and Dzielak, 1986;
Takeichi et al., 1981; Fernandes et al., 1986; Khraibi et al., 1984). Thymic
transplants from neonatal WKY to neonatal SHR will attenuate the
development of hypertension (Norman et al., 1985). Also,
immunosuppressive drugs, such as cyclophosphamide, will attenuate the
development of hypertension in SHR (Khraibi et al., 1984) Immune
system abnormalities, such as increased levels of serum immunoglobulins
and the presence of antibodies directed against self vascular components,
have also been noted in hypertensive human subjects (Svendson, 1979;
Raff and Wortis, 1970; Mathews et al., 1974). These studies looking at

immune factors in the SHR have used adult rats that already had
established hypertension (Norman and Dzielak, 1986; Takeichi et al., 1981;
Fernandes et al., 1986). Therefore, the immune status could have resulted
from the hypertension. The goal of this study was to look for alterations
in lymphocyte populations that occur in the pre-hypertensive as well as
during the hypertensive stages of development of these animals.
Lymphocytes are a major effector cell in the immune system. Besides
being involved in antibody production and direct cell killing, activated
lymphocytes secrete lymphokines, which include various chemical
mediators that are capable of acting locally as well as centrally (Blalock,
1984; Grant et al., 1979a). Any differences in the immune system in the
pre-hypertensive state will provide additional evidence linking immune
system alterations to the development of hypertension. The SHR begins
to develop hypertension at four to five weeks of age. Blood pressure rises
rapidly for the next two to three months and reaches an asymptote at four
months (Figure 4-1). Therefore, we studied rats at two weeks, four weeks,
two, three and four months of age.
There are four major components of the immune system's lymphocyte
populations which can be measured in the rat using specific monoclonal
antibodies. These include the B-cells, the T-cells, and two subpopulations
of T-cells, the T-helper cells and the T-nonhelper cells which include the
cytotoxic T lymphocytes, the suppressor T lymphocytes and some natural
killer (NK) cells. The purpose of this present study is to examine different
lymphocyte populations at various time points during the development
of high blood pressure in the SHR.

Purpose
The purpose of this study was to analyze changes in lymphocyte
populations occurring in the prehypertensive as well as hypertensive
phases of development in the SHR.
Rationale
The immune system, especially those factors which affect the
development of lymphocytes, has been implicated in the development of
hypertension in the SHR. To test if this is the result of hypertension or
appears before hypertension these experiments are undertaken.

Methods
Animals
Two week old, male SH and WKY rats were obtained from breeding
pairs purchased from Charles River Labs, Inc (Wilmington, MA). Male
SHR and WKY rats (n=8/group) used for the remaining time points were
obtained at 28 days of age from Charles River Labs, Inc. A separate group
of five month old, male WKY (n=5) and Sprague-Dawley (n=6) rats was

also obtained from Charles River Labs for comparison of these two

normotensive strains. Upon arrival, all rats were placed on ad-lib rat
chow and water diets in a temperature controlled room with 12:12, light:
dark cycle.
Blood Samples
In order to obtain blood from the two week old animals a thoracotomy
was performed under Metofane (Pitman-Moore, Inc., Washington
Crossing, NJ) anesthesia and as much blood as possible obtained via direct
cardiac puncture. Only animals that produced at least 700ul were used in
this study. The small number of animals (WKY, n=3; SHR, n=4) at the

two week age point is a reflection of the difficulty involved in obtaining
sufficient quantities of blood from such young animals.
At 34 days of age and at four week intervals thereafter blood was
obtained from the remaining animals. The same group of SHR and WKY
animals was used throughout the remainder of the study. Blood was
obtained from these animals as described in Chapter 2.
Lymphocyte Preparation
Lymphocytes were separated and prepared for FACS analysis as
described in Chapter 2.
Blood pressure
Systolic blood pressure recordings were made in unanesthetized,
-restrained animals using a tail plethysmograph.
Statistical analysis
All populations were compared using Students t test for comparison of
means. A level of p<0.05 was considered significant.

Results
Lymphocyte populations
In the prehypertensive phase, at two weeks of age, SHR demonstrate a
significant decrease in the percentage of both T-nonhelper cells (14.11.2 vs
18.20.9) and B-cells (35.81.8 vs 39.11.5) compared to WKY rats (p<0.05)
(Figure 4-2). There were no differences in the total T-cell and T-helper cell
populations. At one month of age the SHR animals showed a
significantly lowered T-nonhelper cell percentage (12.63.0 vs 18.93.8)
(p<0.05) (Figure 4-3). No difference was found in B-cells. At two months
of age the T-nonhelper cells were reduced relative to the WKY controls
(23.03.2 vs 29.93.2) and the T-helper population in SHR was

significantly higher (55.61.9 vs 51.42.4) (Figure 4-4). The picture at three
months (Figure 4-5) is similar to the picture at one month with the T-
nonhelper population continuing to be reduced in the SHR (25.50.61 vs
30.42.0). The T-helper cell difference noted at 2 months was no longer
significant. At four months of age we see a much more dramatic change
in the lymphocyte profile (Figure 4-6). The T-nonhelper cells continued to
be significantly decreased (27.71.8 vs 33.42.5) and at this time point all
lymphocyte populations examined were also significantly decreased
relative to the WKY animals (p<0.05).
Figure 4-7 shows a comparison of four month old WKY and Sprague-
Dawley animals. No significant differences were noted in any lymphocyte
population.
Blood pressures
Data was obtained from the SHR and WKY rats at the termination of
the experiment (Table 4-1). The SHR showed a significant increase in
systolic blood pressure (p<0.05).

Discussion
The results show that even before hypertension develops, SHR have a
deficit in T-nonhelper cells. This deficit persists throughout the
development and maintenance of hypertension. The SHR showed
differences in their lymphocyte profile at both 2 weeks and one month of
age relative to their genetic control, the WKY. This is in the
prehypertensive phase of development. Both the two week and four week
old SHR showed a decrease in their T-nonhelper cell population (Figures
4-2 and 4-3). This is in agreement with other studies done on mature SHR
(Norman et al., 1985; Norman and Dzielak, 1986). This T-nonhelper
deficit remained throughout the study period of four months. These

results confirm the finding of Norman and Dzielak (Norman et al., 1985;
Norman and Dzielak, 1986) and demonstrate that the immunologic
dysfunction which they found in SHR is not a secondary adaptation to
hypertension but a pre-existing condition found in the SHR but not in the
WKY and Sprague- Dawley normotensive rats. Therefore the decrease in
the T-nonhelper population which we have found may contribute to the
etiology of hypertension in the SHR. The T-nonhelper cell population
includes cytotoxic T cells, suppressor T-cells, as well as some NK cells.
A major question posed by this finding is whether the impairment in
T-nonhelper cells contributes to hypertension via a direct action of the
immune system on vascular components or indirectly via lymphokines
-and other chemical mediators released from other immunoregulatory cell
types acting on the CNS. The impairment of T-nonhelper cells could
permit the emergence of vascular inflammation leading to the deposition
of atherosclerotic plaque, or the reduction in T-nonhelper cells could be
disturbing the balance of chemical mediators produced by the other cell
types. SHR do exhibit numerous localized areas of vascular inflammation
throughout their cardiovascular system (Tadeichi et al., 1986).
A second question is why the T-nonhelper cells are reduced in the first
place. There are data to indicate that the peptides produced in the brain
can influence the system (Blalock et al., 1985). One possible method of
action is that centrally synthesized peptides, such as CRF, may be acting on
immunocompetent cells directly. The different cell types found in the
immune system have receptors for and can synthesize centrally active
substances (Blalock, 1984; Johnson and Torres, 1985; Blalock et al., 1985).

However, recently, Irwin et al (Irwin et al., 1988), have demonstrated that
CRF acts on the immune system by activating the SNS.
The SHR also demonstrates alterations in the brain renin-angiotensin
system. Specifically, the SHR has shown higher levels of Ang II in the
brain (Phillips and Kimura, 1988). When infused ICV into the brains of
intact normotensive rats, Ang II has the effect of causing an increase in
SNS activity as well as a increase in AVP secretion (Unger et al., 1981).
This mimics the condition in the SHR which also shows increases in both
SNS activity (Norman and Dzielak, 1986; Judy et al., 1976) and AVP
secretion (Crofton et al., 1978). Central Ang II also plays a role in the
release of ACTH. This increase in central Ang II may explain why SHR
-have increased plasma levels of corticosterone (Dietz et al., 1978).
The SNS has been shown to influence the immune system (Irwin et al.,
1988; del Ray et al., 1985; Braun et al., 1985). The evidence suggests that
SNS activity tends to attenuate immune function (Irwin et al., 1988; del
Ray et al., 1985; Braun et al., 1985). It is known that the SNS sends fibers to
immune organs such as the thymus, spleen and lymph nodes (Bulloch,
1985). These fibers tend to innervate areas rich in T-cells but avoid areas
where high B-cell concentrations are found. Given the fact that SHR
exhibit higher SNS activity, as well as other changes in their
catecholamine systems (Borkowski and Quinn, 1984; Kuchel et al., 1987), it
is not surprising that we would find alterations in the percentage of
different lymphocyte populations.
Besides this increase in SNS activity, the SHR also demonstrates an
increase in AVP secretion (Crofton et al., 1978). There are several lines of
evidence that suggest an immunoregulatory role for AVP. Plasma levels

of AVP increase following exposure of rats to bacterial endotoxin. (Kastin,
1986). Receptors for AVP are found on T-cells (Johnson and Torres, 1985)
and these may be influencing lymphokine production and secretion.
Evidence of AVP immunoreactivity is found in important lymphoid
tissues such as lymph nodes (Aravich et al., 1987) and the thymus
(Markwick, 1986). Also, vasopressin deficient rats exhibit increased
natural killer cell activity (Yirmiya et al., 1989). Interestingly, these rats
show a decreased amount of CRF in the brain (Krieger et al., 1977). When
CRF is injected centrally this results in a decrease in natural killer cell
activity (Irwin et al., 1988) that is believed to be brought about by an
increase in SNS activity (Brown et al., 1982). AVP can also potentiate the
release of CRF and ACTH (Gillies at al., 1982; Gonzalez-Luque et al., 1970).
This would impact on the immune system through the effects of
gluccocorticoids. Thus, this increase in AVP secretion found in the SHR
may influence the immune system through a variety of mechanisms.
AVP levels and SNS activity are altered in the SHR. These two factors
have also been shown to have some influence on the immune system.
The immune system itself has been implicated in the pathogenesis of
hypertension in the SHR (Takeichi et al., 1981; Takeichi et al., 1986).
Immunosuppression will attenuate the development of hypertension in
the model (Khraibi et al., 1984). Thymic transplants from WKY to
neonatal SHR will also cause these animals to become less hypertensive
(Norman and Dzielak, 1986). SHR given antithymocyte serum show a
reduction in blood pressure relative to controls (Bendich et al., 1981). It
appears certain that the immune system is at least in part responsible for
the development of hypertension in the SHR.

In the normal situation the balance between the different lymphocyte
populations is carefully controlled. As has been mentioned previously,
lymphocytes are capable of producing chemical mediators. Other
lymphoid cells such as macrophages also secrete chemical mediators. It is
through these messengers that the various cells of the immune system
interact with one another. Because the chemical mediators released by
one cell type may have a profound effect on the proliferation and
differentiation of another cell type, it is possible that very subtle changes in
certain cell types may have large effects throughout the entire immune
system. Both T and B lymphocytes are capable of lymphokine secretion
(Grant et al., 1979a). Included in the lymphokine category are a variety of
-substances including such things as osteoclast activating factors, PMN
migration inhibitory factors, and ACTH-like substances, as well as others
not fully explored (Blalock, 1984; Grant et al., 1979a). These substances are
capable of acting at local sites as well as acting centrally. From this it is
possible to hypothesize that an imbalance in various cells of the immune
system may lead to the over or under production of various lymphokines
that may exert numerous effects through peripheral and central action. In
this particular case a decrease in the amount of suppressor cells may be
releasing the brake on the action and activity of the other cell types. The
resulting release of lymphokines and/or other chemical mediators could
be acting centrally to influence such things as central angiotensin II levels
and sympathetic output. Interestingly, lymphocytes from both
prehypertensive and hypertensive SHR have a lower intracellular pH
than those from WKY (Batlle et al., 1990b). This condition may further
influence the production and secretion of lymphokines from these cells.

This decrease in the amount of suppressor cells may also be influencing
the production of antibodies. The release of the braking effect provided by
the suppressor cells coupled with the imbalance of lymphokine secretion
could increase the production of autoantibodies directed against vascular
components. In human hypertensive patients antibodies of this type have
been identified (Svendson, 1979). The SHR does posses numerous areas of
localized vascular inflammation (Takeichi et al., 1986). In the SHR the
alterations in cell populations became more numerous as the animals
progressed in age.
It has become apparent recently that when comparisons are made
between the SHR and WKY strains, sometimes it may be the WKY
animals that are "abnormal". For this reason, we compared two
normotensive strains, the WKY and Sprague-Dawley. We found no
differences between these strains for any of the lymphocyte populations
examined. These results indicate that it is indeed the SHR that is
"abnormal".

In summary we present data showing alterations in various
lymphocyte populations in both the prehypertensive and hypertensive
phases of development in the SHR. These results support the hypothesis
that immune dysfunction is involved in the pathogenesis of the
hypertensive state in this model.

Figure 4-2: Percentage of various lymphocyte populations at 2 weeks of
age in SHR and WKY animals. = p<0.05 for SHR vs WKY. Values are
Mean SEM.

100

90go [ WKY (n=8)

80 SHR (n=8)

r 70-

60-

450-
40
% 4
0 %

T-cell T-nonhelper T-helper B-cell

Figure 4-3: Differences in lymphocyte populations at four weeks of age in
the genetically hypertensive SHR compared to its normotensive control,
the WKY. = p<0.05 for SHR vs WKY. Values are Mean S.E.M.

CHAPTER 5
GUANETHIDINE TREATMENT ALTERS LYMPHOCYTE POPULATIONS
AND REDUCES BLOOD PRESSURE IN SPONTANEOUSLY
HYPERTENSIVE RATS
Introduction
Several lines of evidence point to the immune system as an important
factor in the etiology of hypertension in the spontaneously hypertensive
rat (SHR) (Takeichi et al., 1981; Norman et al., 1985; Bendich et al., 1981;
Norman and Dzielak, 1986; Khraibi et al., 1984). Thymic implants from
*WKY to neonatal SHR will attenuate the development of hypertension
(Norman et al., 1985; Norman and Dzielak, 1986). Treatment with
immunosupressive drugs will also lower blood pressure in SHR (Khraibi
et al., 1984). In our own lab, we have found alterations in the percentages
of specific lymphocyte populations in SHR from as early as two weeks of
age (Chapter 4). This evidence suggests that the immune system is linked
to the development of hypertension in this model.
Increased sympathetic nervous system (SNS) activity is a well
characterized phenomenon in SHR (Norman and Dzielak, 1986; Judy et

al., 1976). This increased SNS action is one factor that has been definitely
associated to the development of high blood pressure in the SHR.
Increases in SNS activity have also been linked to alterations in immune
system function and components, such as a decrease in natural killer cell
cytotoxicity (Irwin et al., 1988). In addition, regional sympathetic
denervation has been shown to stimulate local immune responses (Braun

et al., 1985). Thus there is evidence to suggest that the sympathetic
nervous system influences the immune system in SHR and contributes to
the development of the genetic expression of hypertension.
The purpose of the present study was examine the effects of peripheral
sympathectomy on various lymphocyte populations and on the
development of hypertension in the SHR. When given to newborn rats,
guanethidine sulfate will produce a peripheral sympathectomy while
having no effect on central noradrenergic neurons (Johnson et al., 1975).
Administration of guanethedine produces a more complete peripheral
sympathectomy than neonatal administration of 6-hydroxydopamine and
does so with no significant effect on central noradrenergic neurons
.(Johnson et al., 1975)
Purpose
The purpose of this experiment was to block the development of the
sympathetic nervous system in SHR and see if this would reverse any of
the alterations in lymphocyte populations previously noted in this strain.
Rationale

The sympathetic nervous system has been shown to exercise control
over the immune system. Spontaneously hypertensive rats have an
overactive sympathetic nervous system therefore blockade of the system
will show if it plays a direct role in the development of lymphocyte
changes and blood pressure simultaneously.

Mass). Untreated male SHR and WKY were obtained from Charles River
Labs, Inc. at 28 days of age and placed in cages with ad-lib rat chow and
water in a room with a 12:12 light:dark cycle.
Guanethidine treatment
Newborn male SHR were given guanethidine sulfate (Sigma Chemical,
St. Louis, MO) at a dose of 100mg/kg/day for the first 25 days of life. The
injections were made subcutaneously in a vehicle of 0.9% NaC1 solution
(10ul/gbwt). The guanethidine solution was made fresh daily and
adjusted to a pH of 7.0-7.4. After weaning these animals were placed in
cages with ad-lib rat chow and water in a room with a 12:12 light:dark
cycle.
,Blood Samples:
Blood was obtained at approximately one, three and four months of age
from each group of animals as described in Chapter 2.
Lymphocyte Preparation
Lymphocytes were separated and prepared for FACS analysis as
described in Chapter 2.
White blood cell (WBC) and lymphocyte counts
Total WBC's were counted directly from whole blood using a Coulter
Counter (S plus IV, Coulter Electronics, Hialeah, FL). The blood is diluted
with an isotonic saline solution. Red blood cells are lysed with a
potassium ferrocyanide solution. The remaining white blood cells are
counted via a voltage resistance method.
Whole blood smears were stained with Wright-Geimsa stain for
lymphocyte determinations. A drop of blood is placed on a coverslip to
produce a blood film. The coverslips are then warm air dried. They are

then placed in an ethanol bath for five minutes followed by placement in
Wright stain for ten minutes. All coverslips are then rinsed with distilled
water. The slips are then placed in Geimsa stain for 15 minutes, rinsed in
distilled water, air dried and mounted. The cells are manually counted.
Blood pressure
Direct arterial pressure measurements were made at five months of age
in all animals. Under metofane anesthesia, a vinyl catheter was ligated in
the left carotid artery. All measurements were recorded after a ten minute
stabilization period.
Statistics:
An analysis of variance followed by a Newman-Kuels test was used to
compare the means of all groups.
Results
Lymphocyte populations
At one month of age, the guanethidine treated SHR had a significantly
higher percentage of T-nonhelper cells than the control untreated SHR
(21.11.0 vs 17.60.4) (p<0.05) (Figure 5-1). There was no significant
difference between the percentage of T-nonhelper cells found in the
guanethidine treated SHR and untreated WKY. Guanethidine treated
SHR showed a significantly higher T-helper percentage (57.80.8 vs
53.41.6) (p<0.05) and a lower B-cell percentage (20.31.2 vs 28.11.7)
(p<0.05) when compared to WKY. Untreated SHR had a lower percentage
of T-nonhelper cells than untreated WKY (17.60.4 vs 22.11.1) (p<0.05).
The results at three months of age are shown in Figure 5-2. The
guanethidine treated SHR have a higher percentage of total T-cells than
untreated SHR (81.22.5 vs 73.41.6) ) (p<0.05). They also possess a higher
percentage of T-helper cells (63.21.4 vs 52.72.1) (p<0.05). The three

month old, guanethidine treated SHR had a lower percentage of B-cells
than either the untreated SHR or WKY controls (17.01.3 vs 21.71.3 and
26.52.3, respectively) (p<0.05). As was the case at one month untreated
SHR had a decreased nonhelper percentage relative to WKY (20.40.6 vs
28.22.8) (p<0.05).
At four months of age (Figure 5-3), untreated SHR had a lower T-
nonhelper percentage than either WKY or guanethidine treated SHR
(14.42.0 vs 23.30.6 or 26.10.5, respectively) (p<0.05). The guanethidine
treated SHR also had a significantly higher T-nonhelper percentage than
the WKY (26.10.5 vs 23.30.6) (p<0.05).
White blood cell and lymphocyte counts
At one month of age there was no significant change in the number of
WBCs between any of the three groups examined (Table 5-1). At four
months of age, WKY had fewer actual numbers of WBCs and lymphocytes
than either untreated SHR or guanethidine treated SHR.
Blood pressure
All blood pressures were recorded vial direct arterial measurements at
five months of age (Table 5-1). The untreated SHR had significantly
higher pressures than either WKY or guanethidine treated SHR
(185.516.4 vs 125.15.8 and 1103.0, respectively) (p<0.05).

Discussion
The results show that sympathectomy at birth by daily treatment with
guanethidine produces SHR rats that do not have high blood pressure as
adults. The results also show that when blood pressure is normalized in
SHR by this method the T-nonhelper cells of the immune system are not

depressed as they are in untreated SHR and follow the same trend as the
T-nonhelper cell numbers in normotensive controls.
The SHR has been shown to possess various immune system
alterations that are related to the development of hypertension (Takeichi
et al., 1981; Norman et al., 1985; Bendich et al., 1981; Norman and Dzielak,
1986; Khraibi et al., 1984). Another well known characteristic of the SHR is
its increased sympathetic nervous system activity (Norman and Dzielak,
1986; Judy et al., 1976). This increase in SNS activity along with an
increase in plasma arginine vasopressin (AVP) are also associated with the
development of hypertension in this model (Norman and Dzielak, 1986;
Judy et al., 1976; Crofton et al., 1978).
The SNS has been shown to influence the immune system (Irwin et al.,
1988; Braun et al., 1985). An increase in SNS activity is associated with a
decrease in immunocompetence or immune system activity. Our
objective was to block peripheral sympathetic activity in the SHR and then
determine if any changes in blood pressure or lymphocyte populations
occurred.
At both one month and four months of age the level of T-nonhelper
cells was significantly greater in the guanethidine treated animals relative
to the control untreated SHR. At four months of age the guanethidine
treated SHR level was also significantly higher than the WKY. This T-
nonhelper cell level appears to be important. SHR have a generalized
depression of T-lymphocytes, especially T-nonhelper lymphocytes
(Takeichi et al., 1981; Norman et al., 1985; Fernandes et al., 1986). In our
lab, this population is depressed as early as two weeks of age, well before
the development of hypertension (Chapter 4). The antibody that we used

here for the T-nonhelper cells labels T-suppressor cells, cytotoxic T-cells as
well as some natural killer cells. The percentages of the different
lymphocyte populations were not reflected in the absolute numbers.
Adult WKY animals had significantly less WBC's and lymphocytes than
either the untreated control SHR or the guanethidine treated SHR. This is
an important observation. It appears to be the relative proportion of one
cell type to another that is important and not the absolute number of cells.
In summary, by blocking peripheral sympathetic output in the SHR we
have demonstrated a decrease in blood pressure and an alteration in the
different lymphocyte populations examined. What appears to be
important here is not the absolute number of cells, but the relative
proportions of one cell type to another. In a normal situation a balance
between the different cell types and their chemical mediators is achieved.
When this balance is disturbed, such as by a decrease in a regulatory cell
type found in the nonhelper population, an imbalance may occur not only
in other cell types but of the chemical mediators they produce. These
chemical mediators include substances that are similar, if not identical to
ACTH, thyroid stimulating hormone (TSH) and leutenizing hormone
(LH) (Naito et al., 1989; Morley et al., 1987; Blalock, 1984). These chemical
mediators may then be acting not only locally in an immunomodulatory
role, but peripherally and even centrally, to influence the body's
homeostatic mechanism.
Guanethidine treatment produces a peripheral sympathetic
denervation (Johnson et al., 1975). The rats treated from birth for 25 days
did not develop hypertension, confirming the important role of the SNS
in the expression of genetic hypertension (Norman and Dzielak, 1986; Judy
et al., 1976). Since the lack of sympathetic activation (or of its overactivity)

64

was associated with the absence of T-nonhelper cell suppression, we can
make two conclusions: a) Changes in the immune system in these SHRs
is mediated by the SNS which implies a central control over the immune
system, and b) T-nonhelper cell suppression and hypertension are related
to the presence of an active SNS. The results are relevant to the concept
that hypertension is expressed when the immune system is suppressed
and the SNS is overactive.

CHAPTER 6
INTERLEUKIN-2 TREATMENT ALTERS LYMPHOCYTE POPULATIONS,
BUT NOT BLOOD PRESSURE, IN SPONTANEOUSLY HYPERTENSIVE
RATS
Introduction
The spontaneously hypertensive rat (SHR) has been given much
attention as a model of human essential hypertension. The exact cause or
causes of the hypertension in this model remain to be fully understood.
Among the factors considered likely are an increase in central Angiotensin
-II (Ang II) (Phillips and Kimura, 1988), an increased sympathetic nervous
system (SNS) activity (Norman and Dzielak, 1986; Judy et al., 1976;
Norman and Dzielak, 1982) and an increase in arginine vasopressin (AVP)
secretion (Crofton et al., 1978). Another factor that has been gaining more
attention in this model is the immune system (Norman and Dzielak, 1986;
Bendich et al., 1981; Takeichi et al., 1986; Norman et al., 1985; Takeichi et
al., 1981). The SHR has been shown to have fewer and less active T-cells
(Takeichi et al., 1981; Takeichi et al., 1986). Thymic implants from WKY
into neonatal SHR can attenuate the hypertensive response (Norman and
Dzielak, 1986; Takeichi et al., 1986; Norman et al., 1985). Also, chronic
immunosuppression with cyclophosphamide attenuates hypertension in
the SHR (Khraibi et al., 1984). Work in our own lab has shown that SHR
exhibit a decrease in the percentage of T-nonhelper cells both in the
prehypertensive and hypertensive phases of development (Chapter 4).

Recently, Tuttle and Boppana (1990) showed that a single bolus
injection of IL-2 given to young SHR could completely normalize blood
pressure for at least two months. IL-2 is a lymphokine that is involved in
the differentiation and proliferation of T-cell subpopulations. Work in
our lab has focused on the balance of the different lymphocyte populations
and how an alteration of this balance may set the stage for the initiation of
various disease processes. Given the fact that we have found alterations
in the percentages of different lymphocyte populations in SHR (Chapter 4)
we decided to repeat the Tuttle and Boppana study and again examine
these same parameters.
Purpose
The purpose of this experiment was to examine the effect of
interleukin-2 on the development of hypertension in SHR. Another goal
was to test the effect of the treatment on the lymphoycte populations that
we have previously examined in this strain.
Rationale
The immune system has been implicated in the pathogenesis of

hypertension in SHR. A previous study showed that treatment with IL-2
could abolish the development of high blood pressure in this model. We
are therefore testing the finding and also testing if the immune response
and blood pressure response to IL-2 are correlated.

Methods
Animals
Twenty-eight day old SHR and WKY were obtained from Charles River
Labs, Inc. (Wilmington, MA). They were housed in wire bottom cages in a

temperature controlled room with a 12 hour light, 12 hour dark cycle. All
animals were given access to ad-lib rat chow and water.
The rats were divided into four groups. SHR were divided into a
control (n=5) or treatment group (n=8, IL-2 treatment). WKY were also
divided into control (n=6) or treatment (n=8, IL-2 treatment) groups.
IL-2 treatment:
Human recombinant IL-2 was purchased from Cell Products, Inc.
(Buffalo, NY) and a working solution was prepared by dilution with
Macrodex 6% in normal saline (Pharmacia Labs, Piscataway, NJ). At 42
days of age all animals that were to recieve IL-2 were individually weighed
and given a single, subcutaneous bolus injection of 5000 units/kg of IL-2.
,Blood preparation

Blood was obtained at approximately three and four months of age
from each group of animals as previously described in Chapter 2.
Lymphocyte Preparation:
Lymphocytes were separated and prepared as described in Chapter 2.
Blood pressure
Systolic blood pressures were obtained in unanesthetized, restrained
animals using a tail plethysmograph. Readings were obtained from all
animals at two months of age and again at four and a half months of age.
White blood cell (WBC) and lymphocyte counts:
Total WBC's were counted directly from whole blood using a Coulter
Counter (S plus IV, Coulter Electronics, Hialeah, FL) and whole blood
smears were stained with Wright-Geimsa for lymphocyte determinations
as described in Chapter 5.

Statistics
The means of all groups were compared using an analysis of variance
followed by a Newman-Kuels test.

Results
Blood pressures and body weight
At two months and four and a half months control SHR and IL-2
treated SHR had significantly increased blood pressure relative to control
WKY and IL-2 treated WKY, respectively (Table 6-1). At neither age were
the blood pressures for the control SHR and IL-2 treated SHR significantly
different.
All rats were weighed at four and a half months. IL-2 treated WKY
weighed more than IL-2 treated SHR (p<0.05). There was no significant
weight difference between the control SHR and WKY or between control
SHR and IL-2 treated SHR.
Lymphocyte populations:
All values are expressed as a percentage of total lymphocytes examined.
At three months of age (Table 6-2) both the control SHR and IL-2 treated
SHR showed a decrease in the T-nonhelper cell population compared to
the control WKY and IL-2 treated WKY, respectively (p<0.05). IL-2 treated
SHR also showed a decrease in the percentage of B-cells present when
compared to IL-2 WKY (p<0.05). The IL-2 treated SHR had a significant
decrease in the percentage of B-cells when compared to control SHR
(p<0.05).
At four month of age (Table 6-3) the picture changes somewhat. The
control SHR animals show a decreased percentage of T-nonhelper cells
relative to the WKY controls (p<0.05). The IL-2 treated SHR also continue
to show a decrease in the T-nonhelper population percentage in relation

to the IL-2 treated WKY (p<0.05). The IL-2 treated SHR showed an increase
in the percentage of T-nonhelper cells and a decrease in the percentage of
T-helper cells in relation to the control SHR (p<0.05). IL-2 treated SHR
retained the decrease in the B-cell percentage compared to the control SHR
that was seen at 3 months of age. IL-2 treated WKY also had a decrease in
the T-helper population relative to control WKY (p<0.05).
White blood cell and lymphocyte counts
At four months of age the control SHR and IL-2 treated SHR had
significantly greater numbers of WBCs and lymphocytes than the control
WKY and IL-2 treated WKY (Table 6-4). There was no significant
difference between the IL-2 treated animals and the control animals for
-either SHR or WKY.
Discussion
Tuttle and Boppana (1990) recently reported that a single bolus injection
of IL-2 in young SHR could abolish the development of hypertension.
The results that we present here do not support this finding. We found
no difference in the blood pressure of SHR treated with a single dose of IL-
2 compared to untreated SHR at two months or four and a half months of
age. IL-2 does have effects on the immune system of the SHR.
IL-2 is a lymphokine that has important functions in the immune
response. It is secreted by T-lymphocytes, especially stimulated T-helper
lymphocytes. Once secreted it can act on other T-lymphocytes as well as B-
lymphocytes to increase proliferation and differentiation. Evidence is
emerging to indicate that IL-2 has other non-immune system related
functions. Peripherally, IL-2 has been shown to have a positive inotropic
effect on isolated rat atria (Elizalde de Bracco et al., 1989). Central nervous

system effects have been observed as well. When injected
intracerebroventricularly, IL-2 causes behavioral sedation in rats. This
effect is blocked by naloxone, indicating that it is mediated via opiate
receptors (De Sarro et al., 1990). There is evidence that IL-2 can increase
the number of neonatal rat sympathetic neurons in culture and that
sympathetic neurons may have IL-2 receptors (Haugen and Letourneau,
1990). IL-2 binding sites have been found in the rat hippocampus, and
these binding sites may be important modulators of cholinergic activity
and release (Araujo et al., 1989a). IL-2 like material has also been isolated
from the rat hippocampus, striatum and frontal cortex (Araujo et al.,
1989b). Significantly, IL-2 has been shown to cause ACTH release from
*pituitary cells and to raise plasma ACTH (Smith et al., 1989; Naito et al.,
1989). Studies in humans have also pointed to a central role for IL-2.
Cancer patients given IL-2 show behavioral and neurologic changes
(Denicoff et al., 1987; Kolitz et al., 1988; Kakumu et al., 1988). Patients with
progressive multiple sclerosis and chronic fatigue syndrome show similar
neuropsychiatric effects as those cancer patients on IL-2 therapy. Current
studies have shown that these patients have higher than normal values of
serum IL-2 (Trotter et al., 1988; Cheney et al., 1989). Even though the
previously mentioned data suggests an important role for IL-2, its exact
function in maintaining normal body homeostasis is unknown.
Studies from our lab have shown that the SHR exhibit decreases in the
percentage of T-nonhelper cells from a very early age through the
development and establishment of hypertension (Chapter 4). Our present
study confirms with this observation. Both the three and four month old
control SHR and IL-2 treated SHR showed decreases in the percentage of

T-nonhelper cells relative to WKY and IL-2 treated WKY, respectively
(Tables 6-2 and 6-3). At four months of age the IL-2 treated SHR also
showed a significant decrease in the percentage of B-cells (Table 6-3) also in
agreement with our previous study (Chapter 4).
Our IL-2 treated SHR showed no significant decrease in blood pressure
relative to untreated SHR. This is in direct conflict with the results of
Tuttle and Boppana (1990). We used the same age of rats, dose of IL-2 and
measured BP for the same time period. Hypertension developed equally
in the two SHR groups. Our rats were obtained from a different source
that those used in the Tuttle and Boppana (1990) study. This could
account for some of the observed differences including the failure of our
SHR to have an attenuated blood pressure response following IL-2
administration. The IL-2 was active because we observed differences in
the lymphocyte populations we examined between the IL-2 treated SHR
and the untreated, control SHR. At three months of age, the IL-2 treated
SHR showed a significant decrease in the number of B-cells. At 4 months
of age this difference was maintained along with a decrease in the
percentage of T-helper cells and an increase in the number of T-nonhelper
cells. It is possible, however, that the IL-2 used in these experiments was
less active than that used by Tuttle and Boppana (1990)
The T-nonhelper population appears to be important for the expression
of hypertension. As mentioned previously, studies in our lab show this
cell population is depressed as early as two weeks of age in SHR (Chapter
4). This finding may be generalizable as this cell population is also
decreased in inbred prehypertensive Dahl salt-sensitive rats (Chapter 8).
The nonhelper antibody that we have used here labels cytotoxic T-
lymphocytes, suppressor T-lymphocytes as well as some natural killer

cells. A reduction in the activity of T-suppressor cells is often associated
with autoimmune disease (Eisen, 1980). SHR have been shown to have
reduced T-lymphocyte function as well as reduced thymus weight
(Takeichi et al., 1986; Takeichi et al., 1981). The present findings and those
of others point to an altered immune system being at least a partial cause
of the development of hypertension in the SHR (Bendich et al., 1981;
Takeichi et al., 1986; Norman et al., 1985; Takeichi et al., 1981; Khraibi et al.,
1984).
Our study does not offer any evidence as to the cause of the observed
alteration in the different lymphocyte populations. However, IL-2 actions
on CNS development and output and IL-2 action on adrenal steroid
production and secretion are two possibilities.
The data presented show that both groups of SHR exhibited increased
numbers of lymphocytes and WBCs. Previous studies in this area are
somewhat confusing. One study showed SHR as having a decrease in the
amount of WBCs (Tadeichi et al., 1981). Our data agrees with a later study

showing SHR having an increase in the number of WBC's (Takeichi et al.,
1986). Both of these previous studies show the amount of lymphocytes to
be statistically unchanged in the SHR. It is difficult to speculate on the
contradictory results found in our study and the two previously
mentioned studies.
This raises an important question. Is it the absolute number of
lymphocytes that is important? It is more likely that the major factor
involved is the proportion of one cell type to another. In a normal
situation the cells of the immune system communicate with each other
via their respective cytokines. In this case a balance is achieved. If the

77

proportions of the different cell types are altered in some way the balance
of chemical mediators can be altered. As has been suggested these
cytokines can have important non-immunologic roles and may be
influencing the body's homeostatic regulating system.
In summary, we present data showing that a bolus IL-2 injection at 42
days of age did not attenuate the development of hypertension in SHR but
did affect the dynamics of the different lymphocyte populations observed.

0

0

u

0
N3

K

to

,6
w

0

)
t=)

I)

. *
vis
Lo o cQ
p C>(f cr cri

C-4 00 NO
CO CO
+ *
o '-: oq N

f) N L O
s o a N

+ *

\ 00 \D 00

I4 CN
in 0N J4

+N

'EU
*sS$

1 (s 5
2 4-4
tth

3 II II
< +

z
0

i

H
w

0

I
^ s
^0
W
Hi
z
U
LZ

+
C C. C.

t-, 6 eH

.ii!i
4
N ad N

t o 10 10

r-4 f -

IS 00 Lr)
> *

(N 0 \0

'L cri eK

LO

--4

6 Vl
II
r lii
CM

*J N

^0 (+1 0
o6o

+
06c00
06
%0 cq 0

0

0

0
Z-

%U

S>4 4

1 11 11.0
V v V v >
+ ^>

fe

z 1

'0

00 to 0o

>C

0
0

0

VH
0

o
So o

81

+
^s0 O CO
\0 ~ if 0T-

0

SSO
CD m
\0 00

>, N C4E
a co to o

-I-

V iV

1 II
" +

CHAPTER 7
ANGIOTENSIN II LEVELS IN THE SPLEENS OF THREE RAT STRAINS
Introduction
Angiotensin II (Ang II) is an important peptide in the hemodynamic
control mechanisms of the body. It is produced in the blood by the
enzymatic cleavage of Angiotensin I by converting enzyme. This
circulating Ang II is a vasoconstrictor and exerts its actions on vascular
beds throughout the body. Besides these direct effects Ang II also
influences blood pressure and fluid balance through potentiating the
effects of norepinephrine and stimulating aldosterone release from the
adrenal cortex (Zimmerman et al., 1987).
Recently, a possible role of Ang II has been suggested in influencing the
immune system. Ang II binding sites have been localized in the rat spleen
as well as in isolated rat and mouse spleen cells (Castren et al., 1987;
Weinstock and Kassab, 1986). Granuloma macrophages in murine
schistosomiasis have been shown to produce Ang II. This Ang II is
chemotactic for T and B lymphocytes (Weinstock and Kassab, 1986).
The spontaneously hypertensive rat (SHR) is an accepted model of
human essential hypertension. Abnormalities in the immune system of
this model have been suggested as a possible cause of the hypertensive
state. Thymic implants from WKY into neonatal SHR will attenuate the
development of hypertension (Norman and Dzielak, 1986; Norman et al.,
1985). SHR given immunosuppressive drugs will also have an attenuated

hypertension (Khraibi et al., 1984). It appears that this model has a
depression of T-lymphocytes, especially the T-suppressor cell population
(Norman et al., 1985; Takeichi et al., 1981). Interestingly, the SHR also
shows an increased level of brain Ang II (Phillips and Kimura, 1988).
The goal of the present study was to look at levels of Ang II in the
spleen, an important immune organ. The spleens of SHR, WKY and
Sprague-Dawley (SD) rat strains were examined. The SHR was chosen
because of the large amount of data concerning immunodeficiency in
these hypertensive animals. The other strains were used as normotensive
controls.
Purpose
The goal of this study was to measure angiotensin II in the spleen of the
rat. A secondary goal was to test if the Ang II levels differed from one
strain to another.
Rationale
Angiotensin II binding has been found in the spleen but no previous

study has measured Ang II in the spleen. The Ang II content of many
tissues differs among different rat strains. Therefore, we predicted that
Ang II would be present in the spleen.

Methods
Animals
Age matched, male SHR, WKY and Sprague Dawley rats were obtained
at four months of age from Charles River Labs, Inc. (Wilmington, MA).
All animals were placed in a temperature controlled room with a 12 hour
light, 12 hour dark cycle. All animals were placed in wire bottom cages,

with access to ad-lib water and rat chow (#5001, Purina Mills, St. Louis,
MO).
Extraction
The rats were anesthetized with Metofane anesthesia (Pitman-Moore)
and decapitated. An incision was made over the left abdominal area and
the spleen dissected out. The spleen was then placed in saline solution
and any excess fat removed. All spleens were then lightly blotted dry,
weighed, cut into three pieces and placed in a mixture of dry ice and
isopentane for snap freezing. All spleens were stored at -80oC.
Individual frozen spleen chunks were weighed and placed in ten
volumes of 1M acetic acid heated to 1250C for 20 minutes, to denature
proteins and prevent hydrolysis. All samples were then homogenized
using an Ultra-Turrax (Tekmar Co., Cincinnati, OH). The homogenate
was then spun at 10,000g for 20 minutes and the supernatant poured off.
The remaining pellet was resuspended in one ml of 1M acetic acid and
centrifuged as before. This supernatant was added to the first supernatant.
SepPak C-18 Purification
For preparation of tissue for RIA and HPLC, SepPak C-18 cartridges were
used. The cartridges were moistened with 3ml of methanol and washed
with ten ml of 1% trifluoroacetic aced (TFA) in water, then coated with
one ml of 1% Polypep solution (Sigma Chemical, St Louis, MO) in water to
protect against nonspecific absorption of angiotensin on the cartridge, and
washed with ten ml of methanol, water, and TFA (80/90/1, vol/vol),
followed by ten ml of 1% TFA in water.
The spleen supernatant was applied to the SepPak cartridge and the
tube was rinsed with ten ml 1% NaCl/1% TFA(vol/vol) which was also
applied. The cartridge was washed twice with five ml of 1% TFA/1% NaCl

(1/1, vol/vol), and two ml of methanol/water/TFA (30/69/1, vol/vol).
Peptides retained in the cartridge were eluted with methanol/water/TFA
(70/29/1, vol/vol). The eluate was dried under air in polypropylene tubes
on a warm plate.
High pressure liquid chromatography (HPLC) and radioimmunoassay
(RIA)
HPLC elutions were performed on spleen samples from SHR and WKY
as well as 3H-AngII for the purpose of confirming the coelution of an Ang
I-like peptide in the spleen with synthetic Ang II. This was accomplished
using a Beckman HPLC model 332 with a method described previously
(Phillips and Stenstrom, 1985).
RIA was performed on all samples for the quantification of Ang II. This
was accomplished using the method of Phillips and Stenstrom (1985).
Statistics
Where two means were being compared Student's t test was used.
When the means from 3 groups were being compared an analysis of
variance followed by a Newman-Kuels test was performed.

Results
Spleen weights
As is shown in Table 7-1, Spleens from the SHR animals weighed
significantly more than the WKY animals (0.6100.047g vs 0.5140.030g).
HPLC elution profile
Figure 7-1 shows that the immunoreactive Ang II found in SHR and
WKY spleens coelutes in the same fraction as that for 3H-Ang II.

Standard curve for RIA
To make sure that there was no non-specific interference from the
spleen tissue, a SepPak purified WKY spleen extract was serially diluted
and compared to the Ang II standard curve (Figure 7-2). The slope of the
sample is parallel to that of the standard curve indicating no non-specific
interference.
Levels of Ang II in different rat strains
Figure 7-3 shows the results of quantifying the Ang II levels from the
spleens of SHR, WKY and Sprague-Dawley rats. Interestingly, the WKY
had significantly less Ang II/g tissue than either the SHR or Sprague-
Dawley (505.224 pg Ang II/g tissue versus 734.978 and 854.068,
respectively) (p<0.05).
Discussion
The results presented here demonstrate that there are high levels of
Ang II in the rat spleen. The Ang II that was extracted from the rat spleen
elutes in the same fraction as that for synthetic Ang II. Control tubes that
contained no tissue showed no evidence of Ang II.
Not only was Ang II found in the spleen, but the levels, in the
hundreds of picograms could not be accounted for merely by the presence
of blood in the tissue. Typical plasma levels of Ang II are in the range of
1OOpg/ml (Van Eekelen and Phillips, 1988). Clearly, these high levels are
not just a reflection of plasma Ang II flowing through the spleen tissue.
The spleen is an important immune organ. It has dense populations of
macrophages as well as T and B lymphocytes. Although the white blood
cell (WBC) arrangement in the spleen is a very dynamic thing, the
different cell types (macrophages, T-lymphocytes and B-lymphocytes) do

tend to be found in generally specific locations within the splenic
structure.
It has been shown that macrophages can produce Ang II. This Ang II is
chemotactic for both T and B lymphocytes (Weinstock and Kassab, 1986). It
is further suggested that this chemotactic activity is mediated via high
affinity Ang II receptors (Weinstock and Kassab, 1986). This fits in with
studies done by other investigators using autoradigraphy who have
shown the presence of Ang II binding sites in rat spleen sections as well as
in isolated rat spleen cells (Castren et al., 1987).
The SHR is often used as a model of human essential hypertension.
Several investigators have linked abnormalities of the immune system to
the development of this hypertensive state. SHR given the
immunosuppressive drug, cyclophosphamide, show an attenuated
development of hypertension (Khraibi et al., 1984). Neonatal SHR
implanted with WKY thymic tissue also show an attenuated hypertensive
response (Norman and Dzielak, 1986; Norman et al., 1985). Finally, SHR
show an immunological depression, suggestive of impaired T-
lymphocyte activity (Takeichi et al., 1981).
The SHR also shows an increased sympathetic nervous system activity
(Judy et al., 1976). The norepinephrine content per gram of spleen tissue is
higher in young SHR, possibly due to an increased sympathetic
innervation (Donohue et al., 1988). This also might partially explain the
decrease in blood flow rate to the spleen of SHR (Kimura et al., 1988).
Our study found an increased Ang II content in the spleens of SHR
versus WKY. This fact may help explain the above observations. Ang H
potentiates the release and action of norepinephrine from sympathetic
nerve terminals (Zimmerman et al., 1987). The WKY spleen Ang II

content was also decreased relative to the other normotensive control, the
Sprague-Dawley rat. It is difficult to speculate on the reason behind this
finding, although variations among strains are sure to play at least some
role. This also brings up the possibility that these relatively low levels of
Ang II in the spleens of WKY are just a unique characteristic of this strain.
This would imply that the levels found in SHR and Sprague-Dawley rats
are "normal".
We have previously shown that the SHR exhibits a decrease in the
percentage of T-nonhelper lymphocytes from as early as two weeks of age
(Chapter 4). Other investigators, as well, have noted a T-cell depression,
especially of T-suppressor lymphocytes in the SHR (Norman et al., 1985;
Takeichi et al., 1981). Increases in SNS activity have been linked to
decreased in immune activity (Irwin et al., 1988; Braun et al., 1985). Even
with these findings we have also found an increase in total WBCs and
lymphocytes in SHR relative to WKY (Chapter 5). This may partially
explain the increased spleen weight we noted in SHR. It is possible to
hypothesize that increased proliferation of some immunocompetent cell
types, brought about by a reduction in the T-suppressor cell population, for
example, may be causing this increase in spleen weight.
In summary, we have shown significant quantities of Ang II in the rat
spleen. Furthermore, these amounts were significantly higher in SHR
and SD rats than WKY animals. Whether this increase in SHR is due to
immune system dysfunction in these animals remains to be
demonstrated. However, it is reasonable to assume that this splenic Ang II
is related to immune system function and that this interaction may be in
some part responsible for the development of the hypertensive state.

Figure 7-1: HPLC elution profile of an Ang II like peptide in the spleens of
a SHR and WKY rat. The fraction at which 3H-Ang II eluted is also
indicated. This figure demonstrates tat the Ang I1-like material coeluted
with synthetic Ang II.

Figure 7-2: Standard curve for Ang II RIA. To make sure there was no
nonspecific interference from the spleen tissue a purified extract was
serially diluted and the slope of the sample was compared to that of the
standard curve. Serial dilution of the sample gives a slope parallel to that
of the sample, indicating no interference. N = 100ul of purified spleen
extract.

CHAPTER 8
ALTERATIONS IN LYMPHOCYTE POPULATIONS OCCUR PRIOR TO
THE DEVELOPMENT OF HYPERTENSION IN INBRED DAHL RATS
Introduction
The Dahl strain of rat is a commonly used model of hypertension.
When fed a high sodium diet Dahl salt sensitive (DS) rats become
hypertensive, but Dahl salt resistant (DR) rats do not. The original strain
developed by L.K. Dahl was not an inbred strain and this has produced a
certain amount of confusion concerning the role of this animal as an
adequate model of hypertension. This problem was overcome by the
development of an inbred strain of Dahl S and R rats from the original
lines by Dr. J. P. Rapp (Rapp and Dene, 1985). These strains are referred to
as S/JR and R/JR for those rats that become rapidly hypertensive when
exposed to a high sodium diet and those that do not, respectively. Both
the inbred and outbred strains share similar characteristics.
Although many characteristics have been looked at in these animals,
little attention has been focused on their immune systems. The immune
system has been implicated in the pathogenesis of hypertension in
humans (Svendson, 1979; Raff and Wortis, 1970; Mathews et al., 1974).
There is also some provocative data showing that immune abnormalities
are at least in part responsible for the development of hypertension in the
spontaneously hypertensive rat (SHR), another established model of
hypertension (Norman et al., 1985; Norman and Dzielak, 1986; Khraibi et
al., 1984). Data from our lab indicate that the SHR demonstrates